Structural and functional nuclear organization of the beta-globin gene locus in vivo.

Lead Research Organisation: Babraham Institute
Department Name: Epigenetics

Abstract

The DNA sequence of the human genome has provided a tremendous insight into our genetic make-up. However the DNA sequence alone has provided few clues as to how genetic information is controlled so that the correct genes are activated at the right time during development and in the proper tissues. We have found that gene regulation is extremely complex with multiple levels of control. Cells package inactive genomic regions into compact structures within the nucleus that may play a role in keeping the genes in those regions silent. Active genes on the other hand are relatively decondensed and highly mobile within the confines of a defined nuclear space. We have shown that individual genes communicate with distal genomic sequence that contain important regulatory information by direct physical contact. Futhermore we have shown that active genes often congregate in discrete nuclear locations that seem to be important for their expression. We will investigate the events necessary for the communication between genes and distal regulatory sequences and the congregation or organization of active genes in the nucleus. This information is vital to understand normal gene function as well as what goes wrong in cases were genes are deregulated such as in cancer.

Technical Summary

The proposed experiments will investigate the higher-order structure (chromosome folding) and functional nuclear organization (placement in the nucleus) of genes during transcriptional activity and inactivity. We have found that widely separated genes on the same chromosome often co-localize when they are transcriptionally active, at frequencies much higher than would be expected by random association. Taken together with published reports, which show high chromatin mobility, individual genes adopting distinct nuclear localizations in association with transcriptional activity and nuclear sub-compartments rich in transcriptional apparatus, these data suggest that gene activity is controlled in part by nuclear organization. We have shown that the beta-globin genes and the distal beta-globin locus control region physically associate in vivo during the process of transcription. This higher-order structure appears to be dependent on modifications to the histone tails of chromatin in sub-domains encompassing the developmentally specific genes. These modifications in turn are dependent on intergenic transcription through the subdomain region suggesting that the process of transcription or the presence of the non-coding RNA targets remodelling and histone modification factors to the sub-domains. However another non-exclusive hypothesis is that intergenic transcription itself is involved in the assembly of higher-order structures by acting as a molecular motor to pull distal regions into functional complexes. We will investigate these models and attempt to determine a hierarchy of events that take place in the normal control of gene expression. We will use state-of-the-art techniques such as RNA TRAP, 3C, and 3D confocal RNA and DNA FISH to assess higher-order chromatin structures as well as coordinate nuclear organization of genes as far apart as 40 Mb. In addition we will develop a high-throughput assay to rapidly build up information about the functional organization of the nucleus from numerous genomic perspectives. This information is vital to understanding normal gene function as well as in cases were genes are deregulated as in cancer.
 
Description BBSRC Project Grant
Amount £450,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2006 
End 03/2009
 
Description BBSRC Project Grant
Amount £380,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2007 
End 03/2010
 
Description EU FP7
Amount £1,000,000 (GBP)
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 01/2011 
End 12/2015
 
Description MRC Project Grant
Amount £380,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 11/2008 
End 10/2011
 
Title Array and sequence data 
Description Genome wide microarray data using e4C and RNA polII ChIPseq data 
Type Of Material Database/Collection of Data/Biological Samples 
Year Produced 2009 
Provided To Others? Yes  
Impact publication in press 
 
Title Transcription factory model 
Description Greatly improved understanding of transcriptional organization in mammalian cells 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2006 
Provided To Others? Yes  
Impact Advance of the field 
 
Title enhanced 4C (e4C) 
Description Imporoved 4C technique 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact paper in press 
 
Description Cohesin collaboration 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided technical expertise and training
Impact 19458616
Start Year 2008
 
Description Goren 
Organisation Hebrew University of Jerusalem
Country Israel 
Sector Academic/University 
PI Contribution Contributed transgenic mice and gene expression data
Impact 17183675
 
Description Highthroughput DNA sequencing of e4C and ChIP material 
Organisation Agency for Science, Technology and Research (A*STAR)
Department Genome Institute of Singapore
Country Singapore 
Sector Academic/University 
PI Contribution We provided material for them to sequence. We then analyzed the results.
Collaborator Contribution Performed high throughput DNA sequencing for us.
Impact Schoenfelder S, Sexton T, Chakalova L, Cope NF, Horton A, Andrews S, Kurukuti S, Mitchell JA, Umlauf D, Dimitrova DS, Eskiw CH, Luo Y, Wei CL, Ruan Y, Bieker JJ, Fraser P. Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells. Nat Genet. 2010 Jan;42(1):53-61.
Start Year 2006
 
Description Intergenic paper 
Organisation University of Chicago
Country United States 
Sector Academic/University 
PI Contribution We performed the experiments and wrote the paper
Collaborator Contribution Provided transgenic mice
Impact 17637845
 
Description Myc paper 
Organisation Babraham Institute
Country United Kingdom 
Sector Private 
PI Contribution We did 95% of the work and wrote the paper
Collaborator Contribution Supplied us with DNA probes and expertise.
Impact 17622196
 
Description NSMB Review 
Organisation Friedrich Miescher Institute for Biomedical Research (FMI)
Country Switzerland 
Sector Academic/University 
PI Contribution Co-authored review
Impact 17984967
Start Year 2006
 
Description Nature Review 
Organisation Medical Research Council (MRC)
Department MRC Human Genetics Unit
Country United Kingdom 
Sector Public 
PI Contribution Co-authored a review
Impact 17522674
Start Year 2007
 
Description Review 
Organisation University of Michigan
Country United States 
Sector Academic/University 
PI Contribution Co-authored a review with Doug Engel
Impact 16751176
Start Year 2006
 
Description BBC news website 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Interviewed and quoted on BBC News webite

none
Year(s) Of Engagement Activity 2008
 
Description BBC radio interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Radio interview on local radio

none
Year(s) Of Engagement Activity 2009
 
Description Judge of Science competition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact Judge student projects in a one day science competition involving 5 different colleges.

none
Year(s) Of Engagement Activity 2009
 
Description Researcher in residence 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact Visit to schools

none
Year(s) Of Engagement Activity 2007,2008
 
Description School visit SVC 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact Lectures at local Village College

Student spent two weeks in the lab as part of work experience
Year(s) Of Engagement Activity 2009,2010
 
Description Schools day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact Hosted students for day of experiments

none
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010